Cell Signalling - Angeliki Malliri

Angeliki completed her undergraduate degree in Biology in the University of Patras in Greece and obtained her PhD from the University of Crete, Greece. She then worked as a postdoctoral scientist in the laboratory of Professor Brad Ozanne in the Beatson Institute for Cancer Research in Glasgow and in the laboratory of Dr John Collard in the Cell Biology Division of the Netherlands Cancer Institute in Amsterdam, The Netherlands, where she was funded by a Marie Curie European Fellowship. In 2004, Angeliki joined the CRUK Manchester Institute and is currently a Senior Group Leader. She was awarded a Professorship in Cell Biology at The University of Manchester in 2018.

Introduction

Neoplasia is driven by deregulated intracellular signalling. Mutations and overexpression in human tumours and cell lines implicate the small GTPase RAC and its activators, the guanine nucleotide exchange factors (GEFs), in the formation and dissemination of a range of cancers. Furthermore, the effects of ablating genes encoding RAC proteins or RAC GEFs in mouse or of pharmacologically inhibiting RAC-GEF/RAC interactions strongly suggest that targeting RAC signalling could constitute an effective cancer treatment. Owing to multiple physiological roles of RAC and moreover RAC functions that antagonise tumour dissemination, sustained suppression of RAC signalling could however be detrimental.

Given this challenge, the research of the Cell Signalling group aims to distinguish RAC-dependent effects that promote tumour growth and progression from those that antagonise tumour progression so that RAC signalling might ultimately be targeted more effectively. Previously we have found that the RAC GEF TIAM1 is essential for RAS-induced tumourigenesis due to its requirement for cell survival and proliferation. Moreover, we have shown that activation of RAC can promote cell migration and invasion connected to post-translational modifications and its activation by specific GEFs. However, alternative pathways, again regulated by post-translational modifications that we have identified, utilise RAC to strengthen cell-cell adhesion and antagonise migration and invasion. We are currently investigating novel roles of the RAC pathway in controlling migration and invasion and in lung tumourigenesis in vivo.

Overview

 

The main focus of the Cell Signalling group is the identification of therapeutic targets in lung cancer, the most common cause of cancer related deaths worldwide. Lung cancer is divided into non-small cell lung cancer (NSCLC, approximately 85% of cases) and SCLC (~15% of cases). The most common histological subtype of NSCLC is adenocarcinoma, of which the most common driver mutation is KRAS. KRAS mutant lung adenocarcinoma (KRASm-LUAD) and SCLC treatments lag behind other lung cancer types, for which targeted therapies offer additional treatments prolonging patient survival. No approved targeted therapies exist for SCLC, and until very recently, nor did they for KRASm-LUAD. However, recently direct inhibitors of KRASG12C (accounting for ~40% of KRASm-LUAD cases) have been approved, but resistance has been documented in relapsing patients. Other KRASm isoforms lack effective targeted therapies. Therefore, current drug development efforts focus not only on KRAS itself, but also on downstream targets. One such downstream target under investigation in our laboratory is the small GTPase RAC1.

 

RAC is a member of the RHO-like family of GTPases and cycles between a GDP- and a GTP-bound state. When GTP-bound, it interacts with various effector molecules that regulate several cellular processes, including proliferation and migration. Multiple mechanisms control RAC activity, including control of nucleotide binding and hydrolysis by guanine nucleotide exchange factors (GEFs) and GTPase Activating Proteins (GAPs) respectively, regulation of subcellular localisation and modulation of RAC protein levels (reviewed in Porter et al., Small GTPases 2017). Moreover, several studies using recombinant RAC and RAC GEF mice have shown that RAC is required for the formation and growth of tumours. In particular, RAC is required for the formation of KRAS-induced lung tumours in mice. Moreover, the RAC GEF TIAM1 is required for the formation and growth of HRAS-induced skin tumours (Malliri et al., Nature 2002). Interestingly, TIAM1 and its homologue STEF/TIAM2 both contain a RAS-binding domain and are considered RAS effectors.

 

Role of TIAM-RAC1 signalling in lung cancer formation and progression

 

The role of TIAM1 and STEF/TIAM2 in NSCLC formation and progression is currently under investigation using in vitro and in vivo models and updates will be given in subsequent annual reports. SCLC is a highly aggressive malignancy broadly divided into neuroendocrine (NE, >80% of SCLC) and non-NE subtypes. Analysis of expression data from SCLC tumours, patient-derived models and established cell lines, showed that the expression of TIAM1 is associated with a neuroendocrine gene program (STEF/TIAM2 levels were very low in all SCLC tumours/cell lines). Moreover, we showed that TIAM1 depletion or RAC1 inhibition reduces viability and tumorigenicity of SCLC cells by increasing apoptosis associated with conversion of BCL2 from its pro-survival to pro-apoptotic function via BH3 domain exposure. We showed that this conversion is dependent upon cytoplasmic translocation of Nur77, an orphan nuclear receptor. Like in other cell types, TIAM1 is present in the nucleus of SCLC cells, where it interacts with Nur77 sequestering it in SCLC cell nuclei. TIAM1 depletion or RAC1 inhibition promoted Nur77 translocation to the cytoplasm. Importantly, mutant TIAM1 with reduced Nur77 binding failed to suppress apoptosis triggered by TIAM1 depletion. In conclusion, TIAM1-RAC1 signalling promotes SCLC cell survival via Nur77 nuclear sequestration (Payapilly et al (2021). TIAM1-RAC1 promote small-cell lung cancer cell survival through antagonizing Nur77-induced BCL2 conformational change. Cell Reports, 37(6), 109979.).

Figure 1: Model depicting the role of TIAM1 in survival of SCLC cells TIAM1 expression is upregulated in NE SCLC. TIAM1 sequesters Nur77 in the nucleus. Depletion of TIAM1 or inhibition of RAC1 activation by TIAM1 leads to cytoplasmic redistribution of Nur77. In the cytoplasm, Nur77 induces exposure of the BH3 domain of BCL2 promoting apoptosis.

 

Role of RAC1 activators in migration and malignant progression

 

Although RAC seems always to promote tumour formation and growth, it may promote or antagonise malignant progression. There are cases where deletion of RAC GEFs leads to more invasive tumours and there are reports suggesting that reduced RAC activity levels correlate with more aggressive tumours (Porter et al., Small GTPases 2017). In vitro data have shown that activation of RAC may lead to opposing migratory phenotypes raising the possibility that targeting RAC in a clinical setting could exacerbate tumour progression. For these reasons it is important to identify the factors that influence whether RAC activation will promote or inhibit migration. One such factor that we have identified is the GEFs that activate RAC. RAC GEFs are multi-domain proteins with many binding partners. We showed that TIAM1 and another RAC GEF, P-REX1, have diametrically opposite effects on cell migration through RAC in certain epithelial cells and fibroblasts: TIAM1 promotes cell-cell adhesions to oppose cell migration while P-REX1 promotes migration. They perform these contrasting roles in cell migration by selecting RAC effectors (Marei et al., Nat Commun 2016). Over-expression of specific GEFs, which occurs commonly in many cancers, can therefore drive different oncogenic signalling pathways. These data on TIAM1 are consistent with the fact that even though TIAM1 knockout mice were resistant to the formation of RAS-induced tumours, the few tumours which did form were more aggressive (Malliri et al., Nature 2002). This highlights two distinct roles for TIAM1/RAC signalling: stimulating tumour formation but suppressing malignant progression. Early work on TIAM1’s role in suppressing migration and invasion focused on its role in strengthening cell-cell junctions, associated with anti-migratory effects (Malliri et al., J Biol Chem 2004). It was also shown that cell-cell adhesion disassembly and scattering of epithelial cells requires depletion of TIAM1 from cell-cell adhesions (Woodcock et al., Mol Cell 2009; Vaughn et al. Cell Rep 2015).

 

But besides these studies showing that TIAM1 inhibits migration by promoting cell-cell adhesion, we have also identified another mechanism by which TIAM1 hinders migration. TIAM1 localises in the nucleus of several colorectal cancer cell lines and nuclear TIAM1 inhibits their migration via suppressing the interaction of the transcriptional co-activator TAZ with its cognate transcription factor TEAD. Suppression of this interaction by TIAM1 inhibited expression of TAZ/YAP target genes implicated in epithelial-mesenchymal transition and cell migration. Consistent with these in vitro data, we showed by staining a microarray of colorectal cancer biopsies that TIAM1 localised to the nuclei of tumour cells. Moreover, nuclear staining intensity significantly decreased with advancing Dukes stage and patients with high nuclear TIAM1 had significantly better survival than those with low nuclear TIAM1 (Diamantopoulou et al., Cancer Cell 2017).

 

More recently, we have uncovered a new role for TIAM1 in regulating the duplication of centrioles – structures at the core of centrosomes found at the poles of the mitotic spindle. Cells normally duplicate centrioles only once per cell cycle. Centriole overduplication is common in many cancers however, promoting aneuploidy but also increasing invasiveness of tumour cells. We found that TIAM1 localises to centrosomes and that its depletion leads to centriole overduplication, lagging chromosomes at anaphase and aneuploidy (Porter et al., J Cell Sci. 2021), indicating another mechanism by which loss of TIAM1 could promote malignant progression.

 

Apart from these roles of RAC1 activators in antagonising cell migration, RAC1 activators are also associated with promotion of cell migration. Interestingly, activation of RAC by STEF/TIAM2 promotes migration (Rooney et al., EMBO Rep. 2010), something we have seen in all cell types tested. STEF/TIAM2 localises at the nuclear envelope, co-localising with the key perinuclear proteins Nesprin-2G and Non-muscle myosin IIB, where it regulates perinuclear RAC1 activity. Interestingly, STEF depletion reduced apical perinuclear actin cables (thick actin bundles which run over the nucleus, constraining its height and guiding nuclear orientation), increased nuclear height and impaired nuclear re-orientation, which is required for optimal cell migration. Finally, STEF depletion reduced expression of TAZ-regulated genes, indicating an alteration in mechanosensing pathways as a consequence of disruption of the actin cap.

Selected Publications

 

Payapilly A, Guilbert R, Descamps T, White G, Magee P, Zhou C, Kerr A, Simpson KL, Blackhall F, Dive C, Malliri A. (2021)
TIAM1-RAC1 promote small-cell lung cancer cell survival through antagonizing Nur77-induced BCL2 conformational change.
Cell Reports 37(6):109979. PubMed abstract (PMID: 34758330)

Porter AP, Reed H, White GRM, Ogg EL, Whalley HJ, Malliri A. (2021)
The RAC1 activator Tiam1 regulates centriole duplication through controlling PLK4 levels.
Journal of Cell Science 134(7):jcs252502. PubMed abstract (PMID: 33758078)

Porter AP, White GRM, Mack NA, Malliri A. (2019)
The interaction between CASK and the tumour suppressor Dlg1 regulates mitotic spindle orientation in mammalian epithelia.
Journal of Cell Science 132(14) jcs230086. PubMed abstract

Woroniuk A, Porter A, White G, Newman DT, Diamantopoulou Z, Waring T, Rooney C, Strathdee D, Marston DJ, Hahn KM, Sansom OJ, Zech T, Malliri A. (2018)
STEF/TIAM2-mediated Rac1 activity at the nuclear envelope regulates the perinuclear actin cap.
Nature Communications 9(1):2124. PubMed abstract

Diamantopoulou Z, White G, Fadlullah MZH, Dreger M, Pickering K, Maltas J, Ashton G, MacLeod R, Baillie GS, Kouskoff V, Lacaud G, Murray GI, Sansom OJ, Hurlstone AFL, Malliri A. (2017)
TIAM1 antagonizes TAZ/YAP both in the destruction complex in the cytoplasm and in the nucleus to inhibit invasion of intestinal epithelial cells.
Cancer Cell, 31(5):621-634.e6. PubMed abstract

Marei H, Carpy A, Woroniuk A, Vennin C, White G, Timpson P, Macek B and Malliri A. (2016) Differential Rac1 signalling by guanine nucleotide exchange factors implicates FLII in regulating Rac1-driven cell migration.
Nature Communications (7):10664-79. PubMed abstract

Whalley HJ, Porter A, White GRM, Diamantopoulou Z, Castañeda-Saucedo E and Malliri A. (2015) Cdk1 phosphorylation of the Rac activator Tiam1 is required for centrosomal Pak activation and regulation of spindle assembly in mitosis.
Nature Communications (6):7437-51. PubMed abstract

Vaughan L, Tan C, Chapman A, Nonaka D, Mack NA, Smith D, Booton R, Hurlstone AF and Malliri A. (2015)
HUWE1 ubiquitylates and degrades the RAC Activator TIAM1 promoting cell-cell adhesion disassembly, migration, and invasion.
Cell Reports (10):88-102. PubMed abstract

Mack NA, Porter AP, Whalley HJ, Schwarz JP, Jones RC, Khaja AS, Bjartell A, Anderson KI and Malliri A. (2012)
β2-syntrophin and Par-3 promote an apicobasal Rac activity gradient at cell-cell junctions by differentially regulating Tiam1 activity.
Nature Cell Biology (14):1169-80. PubMed abstract

Castillo-Lluva S, Tatham MH, Jones RC, Jaffray EG, Edmondson RD, Hay RT and Malliri A. (2010)
SUMOylation of the GTPase Rac1 is required for optimal cell migration.
Nature Cell Biology (12):1078-85. PubMed abstract

Malliri A, van der Kammen RA, Clark K, van der Valk M, Michiels F and Collard JG. (2002)
Mice deficient in the Rac activator Tiam1 are resistant to Ras-induced skin tumours.
Nature (417):867-71. PubMed abstract

Postdoctoral Fellows
Martin Baker
William McDaid
Leah Wilson

Scientific Officer
Peter Magee

Graduate Students
Lucy Ginn
Ryan Guilbert
Hannah Reed
Joshua Searle
Kirsten Tinsley

Clinical Fellow
George Morrissey